Prospective CCR5 small molecule antagonist compound design using a combined mutagenesis/modeling approach.

The viral resistance of marketed antiviral drugs including the emergence of new viral resistance of the only marketed CCR5 entry inhibitor, maraviroc, makes it necessary to develop new CCR5 allosteric inhibitors. A mutagenesis/modeling approach was used (a) to remove the potential hERG liability in an otherwise very promising series of compounds and (b) to design a new class of compounds with an unique mutant fingerprint profile depending on residues in the N-terminus and the extracellular loop 2. On the basis of residues, which were identified by mutagenesis as key interaction sites, binding modes of compounds were derived and utilized for compound design in a prospective manner. The compounds were then synthesized, and in vitro evaluation not only showed that they had good antiviral potency but also fulfilled the requirement of low hERG inhibition, a criterion necessary because a potential approved drug would be administered chronically. This work utilized an interdisciplinary approach including medicinal chemistry, molecular biology, and computational chemistry merging the structural requirements for potency with the requirements of an acceptable in vitro profile for allosteric CCR5 inhibitors. The obtained mutant fingerprint profiles of CCR5 inhibitors were used to translate the CCR5 allosteric binding site into a general pharmacophore, which can be used for discovering new inhibitors.

Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays.

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure-activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

Alfuzosin delays cardiac repolarization by a novel mechanism.

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD)(60) in rabbit Purkinje fibers (p < 0.05) and QT in isolated rabbit hearts (p < 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericia's formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hK(v)11.1, hK(v)4.3, and hK(v)7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, approximately 30x C(max), alfuzosin significantly increased whole-cell peak sodium (hNa(v)1.5) current (p < 0.05), increased the probability of late hNa(v)1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNa(v)1.5 burst duration and number of openings per burst between 2- and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hK(v)11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.

Curcumin potently blocks Kv1.4 potassium channels.

Curcumin, a major constituent of the spice turmeric, is a nutriceutical compound reported to possess therapeutic properties against a variety of diseases ranging from cancer to cystic fibrosis. In whole-cell patch-clamp experiments on bovine adrenal zona fasciculata (AZF) cells, curcumin reversibly inhibited the Kv1.4K+ current with an IC50 of 4.4 microM and a Hill coefficient of 2.32. Inhibition by curcumin was significantly enhanced by repeated depolarization; however, this agent did not alter the voltage-dependence of steady-state inactivation. Kv1.4 is the first voltage-gated ion channel demonstrated to be inhibited by curcumin. Furthermore, these results identify curcumin as one of the most potent antagonists of these K+ channels identified thus far. It remains to be seen whether any of the therapeutic actions of curcumin might originate with its ability to inhibit Kv1.4 or other voltage-gated K+ channel.

Modulation of native T-type calcium channels by omega-3 fatty acids.

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.

Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms.

Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (V(m)) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5'-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 microM) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells.

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells.

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.